Expression Of Tumor Necrosis Factor Receptor Research Articles

Pentoxifylline attenuates cytokine stress and Fas system in syngeneic liver proteins induced experimental autoimmune hepatitis

BackgroundApoptosis is a hallmark in the pathogenesis of autoimmune hepatitis (AIH). Cytokine stresses and extrinsic apoptotic pathway have been implicated in this type of hepatic injury. Pentoxifylline plays an important role in controlling inflammation and apoptosis in different autoimmune diseases. AimTo assess the protective effect of pentoxifylline for 30days against pro-inflammatory cytokines as tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ) and mediators of extrinsic apoptotic pathway involving TNF receptor 1 (TNFR1) and its ligand TNF-α and Fas receptor and its ligand (FasL) in experimental autoimmune hepatitis (EAH) model. MethodsEAH was induced by intraperitoneal injection of syngeneic liver antigen emulsified in complete Freund's adjuvant (CFA) in male C57BL/6 mice. Five groups of mice were used: two control groups; Control PBS group and Control CFA group, EAH group and two EAH+pentoxifylline treated groups in doses (100 or 200mg/kg/d, given by oral gavage). Serum transaminase, pro-inflammatory cytokines (TNF-α and interferon-γ) and hepatic caspase-8 and 3 activities were evaluated. Signs of autoimmune hepatitis were confirmed by liver histology. In addition, hepatic TNFR1, Fas and FasL mRNA expression were assayed. ResultsSerum transaminase levels and signs of AIH observed in EAH mice were significantly reduced by pentoxifylline. Upregulated serum TNF-α, IFN-γ, hepatic caspase-8 and 3 activities and TNFR1, Fas and FasL mRNA expression in liver tissues in EAH group were significantly downregulated by pentoxifylline. ConclusionPentoxifylline protects against syngeneic liver antigen induced hepatitis and associating apoptosis through attenuating the exaggerated cytokine release and extrinsic apoptotic pathway. Thus, this may represent a new therapeutic strategy for hepatitis.

TNF Signaling in Peritoneal Mesothelial Cells: Pivotal Role of cFLIPL.

♦ BACKGROUND: Peritoneal dialysis (PD) coincides with high concentrations of proinflammatory cytokines, such as tumor necrosis factor (TNF), in the peritoneal cavity. During treatment, chronic inflammatory processes lead to damage of the peritoneal membrane and a subsequent ultrafiltration failure. Human peritoneal mesothelial cells (HPMCs) play a central role as mediators and targets of PD-related inflammatory changes. Although TNF Receptor 1 (TNFR1) is expressed in high numbers on the cells, TNF-induced apoptosis is inhibited. Here, the underlying molecular mechanisms of TNFR1 signaling in HPMCs are investigated. ♦ METHODS: Human peritoneal mesothelial cells were isolated from the omentum of healthy donors and the dialysis solution of PD patients. Flow cytometry was applied to determine cell surface expression of TNFR1 on HPMCS from healthy donors in absence or presence of TNF or PD fluid (PDF) and were compared to TNFR1 expression on cells from PD patients. To investigate TNFR1-mediated signaling, HPMCs were treated with PDF or TNF, and expression patterns of proteins involved in the TNFR1 signaling pathway were assessed by western blot. ♦ RESULTS: Incubation with PDF led to a significant up-regulation of TNFR1 on the cell surface correlating with elevated TNFR1 numbers on HPMCs from PD patients. Investigations of underlying molecular mechanisms of TNFR1 signaling showed that PDF affects TNFR1 signaling at the proapoptotic signaling pathway by upregulation of IκBα and downregulation of cFLIPL. In contrast, TNF exclusively induces the activation of NFκB by an increase of phosphorylated IκBα. ♦ CONCLUSIONS: Novel and relevant insights into the mechanisms of TNFR1-mediated signaling in HPMCs with an impact on our understanding of PD-associated damage of the peritoneal membrane are shown.

The influence of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2.

Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.

Tripartite motif-containing 28 bridges endothelial inflammation and angiogenic activity by retaining expression of TNFR-1 and -2 and VEGFR2 in endothelial cells.

Angiogenesis and inflammation are regarded as important factors in the pathogenesis of chronic inflammation, cancer, and wound healing. Recent studies have supported prior evidence that common signaling pathways are involved in angiogenesis and inflammatory responses; however, key factors controlling both processes remain unclear. Although tripartite motif-containing (TRIM)-28 is known to have an immunosuppressive role in immune cells, its expression level and role in endothelial cells (ECs) are still unclear. In this study, we investigated the role of TRIM28 in inflammatory responses and angiogenic activity of ECs for the first time. We showed that TRIM28 is the most abundant TRIM family member and is localized in nuclei of ECs. Small interfering RNA-mediated knockdown of TRIM28 strikingly suppressed expression of TNF receptor (TNFR)-1 and -2, decreased TNF-α-induced phosphorylation of IKKα/β and IκBα and degradation of IκBα and nuclear translocation of p65, and suppressed basal level and TNF-α-induced expression of chemokines and adhesion molecules, including VCAM-1, IL-6, ICAM-1, E-selectin, and monocyte chemoattractant protein (MCP)-1. Unexpectedly, IL-8 was potentiated by TRIM28 knockdown in ECs in an NF-κB-inducing kinase-dependent manner. Meanwhile, knockdown of TRIM28 inhibited expression of VEGF receptor 2 and suppressed VEGF-induced proliferation and tube formation by ECs. Finally, knockdown of TRIM28 suppressed recruitment of ECs in vivo in a murine synthetic basement membrane model. In summary, we found that TRIM28 acts as a central factor in controlling endothelial inflammatory responses and angiogenic activities by retaining expression of TNFR-1 and -2 and VEGF receptor 2 in ECs.-Wang, Y., Li, J., Huang Y., Dai, X., Liu, Y., Liu, Z., Wang, Y., Wang, N., Zhang, P. Tripartite motif-containing 28 bridges endothelial inflammation and angiogenic activity by retaining expression of TNFR1 and -2 and VEGFR2 in endothelial cells.

Deep brain stimulation of the anterior nucleus of the thalamus reverses the gene expression of cytokines and their receptors as well as neuronal degeneration in epileptic rats

BackgroundDeep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) is effective in seizure control. However, the mechanisms remain unclear. MethodsSixty-four rats were randomly assigned to the control group, the kainic acid (KA) group, the sham-DBS group and the DBS group. Video-electroencephalogram (EEG) was used to monitor seizures. Quantitative real time PCR (qPCR) was applied for detecting interleukin-1 beta (IL-1β), IL-1 receptor (IL-1R), IL-6, IL-6 receptor (IL-6R), gp130, tumor necrosis factor-alpha (TNF-α), TNF-receptor 1 (TNF-R1) and TNF-receptor 2 (TNF-R2) expression 12h after the establishment of an epileptic model. The neuronal structural degeneration in the hippocampus was evaluated with transmission electron microscopy (TEM) at this same time point. ResultsThe seizure frequency was 48.6% lower in the DBS group compared with the sham-DBS group (P<0.01). The expression of IL-1β, IL-1R, IL-6, IL-6R, gp130, TNF-α and TNF-R1 was elevated in both the KA and the sham group compared with the control group (all Ps<0.01). Additionally, ANT-DBS was able to reverse this gene expression pattern in the DBS group compared with the sham-DBS group (all Ps<0.01). There was no significant difference in TNF-R2 expression among the four groups. The neuronal structural degeneration in the KA group and the sham-DBS group was more severe than that in the control group (injury scores, all Ps<0.01). ANT-DBS was also capable of relieving the degeneration compared with the sham-DBS group (injury score, P<0.01). ConclusionsThis study demonstrated that ANT-DBS can reduce seizure frequency in the early stage in epileptic rats as well as relieve the pro-inflammatory state and neuronal injury, which may be one of the most effective mechanisms of ANT-DBS against epileptogenesis.

Green tea extract treatment reduces NFκB activation in mice with diet-induced nonalcoholic steatohepatitis by lowering TNFR1 and TLR4 expression and ligand availability

NFκB-mediated inflammation contributes to liver injury during nonalcoholic steatohepatitis (NASH). We hypothesized that antiinflammatory activities of green tea extract (GTE) during NASH would lower tumor necrosis factor receptor-1 (TNFR1)- and Toll-like receptor-4 (TLR4)-mediated NFκB activation. Male C57BL6/J mice (6 weeks old) were fed a low-fat (LF) or high-fat (HF) diet for 12 weeks to induce NASH. They were then randomized to continue on these diets supplemented with 0 or 2% GTE (n=10/group) for an additional 8 weeks prior to evaluating NASH, NFκB inflammation and TNFR1 and TLR4 receptor complexes and their respective ligands, TNFα and endotoxin. HF feeding increased (P<.05) serum alanine aminotransferase (ALT) activity and histological evidence of NASH compared with LF controls. HF-mediated increases in NFκB p65 phosphorylation were also accompanied by increased serum TNFα and endotoxin concentrations, mRNA expression of hepatic TNFR1 and TLR4 and MyD88 protein levels. GTE in LF mice had no effect (P>.05) on liver histology or inflammatory responses. However, GTE in HF mice decreased biochemical and histological parameters of NASH and lowered hepatic p65 phosphorylation in association with decreased serum TNFα, mRNA expression of TNFR1 and TLR4 and MyD88 protein. GTE in HF-fed mice also lowered serum endotoxin and up-regulated mRNA expression of duodenal occludin and zonula occluden-1 and ileal occludin and claudin-1 that were otherwise lowered in expression by HF feeding. These data suggest that dietary GTE treatment reduces hepatic inflammation in NASH by decreasing proinflammatory signaling through TNFR1 and TLR4 that otherwise increases NFκB activation and liver injury.

Expression of tumor necrosis factor receptor associated factor 6 in human esophageal carcinoma and its clinical significance

Objective To investigate the expression and clinical significance of tumor necrosis factor receptor associated factor 6(TRAF6) in human esophageal cancer. Methods The clinical data of 72 patients with esophageal cancer were collected.Immunohistochemistry method was used to determine TRAF6 expression in esophageal carcinoma and its adjacent normal tissue, and its relationship with clinical pathological features was explored. Results The TRAF6 positive expression rate in esophageal cancer tissue was 66.13%, which was significantly higher than that of normal tissue (13.89%), the difference between the two groups was statistically significant(χ2=56.850, P<0.01). And TRAF6 expression level was significantly correlated with esophageal cancer clinical staging, lymph node metastasis(χ2=6.818, 4.428, all P<0.05), but TRAF6 expression was not correlated with age, sex, tumor differentiation. Conclusion The expression level of TRAF6 in esophageal carcinoma was significantly increased, and there was a significant correlation between the TRAF6 expression level and clinical pathological characteristics. Key words: Esophageal neoplasms; Tumor necrosis factor receptor-associated peptides and proteins; Immunohistochemistry

A new form of amphotericin B - the complex with copper (II) ions - downregulates sTNFR1 shedding and changes the activity of genes involved in TNF-induced pathways: AmB-Cu2+ downregulates sTNFR1 shedding and changes the activity of genes involved in TNF-induced pathways.

A new form of amphotericin B (AmB)- complex with copper (II) ions (AmB-Cu2+) - is less toxic to human renal cells. Cytokines, including Tumor Necrosis Factor (TNF), are responsible for nephrotoxicity observed in patients treated with AmB. Another problem during therapy is the occurrence of oxidized forms of AmB (AmB-ox) in patients' circulation. To elucidate the molecular mechanism responsible for the reduction of the toxicity of AmB-Cu2+, we evaluated the expression of genes encoding TNF and its receptors alongside encoding proteins involved in TNF-induced signalization. Renal cells (RPTECs) were treated with AmB, AmB-Cu2+ or AmB-ox. The expression of TNF and its receptors was evaluated by ELISA tests and real-time RT-qPCR. The expression of TNF-related genes was appointed using oligonucleotide microarrays. Only sTNFR1 was detected, and its level was lower in AmB-Cu2+- and AmB-ox-treated cells. TNFR1 mRNA was downregulated in AmB-ox, while TNFR2 mRNA was upregulated in AmB and AmB-Cu2+. Several changes in the expression of TNF-related genes coincided with changes in the expression of TNF receptors. The lower toxicity of AmB-Cu2+ could result from the changes in the expression of TNF receptors, which coincided with the changes in the expression of genes encoding proteins involved in TNF-induced pathways. This situation might subsequently result in a changes in intracellular signalization and influence the toxicity of tested forms of AmB on renal cells.

Targeted genomic analysis reveals widespread autoimmune disease association with regulatory variants in the TNF superfamily cytokine signalling network.

BackgroundTumour necrosis factor (TNF) superfamily cytokines and their receptors regulate diverse immune system functions through a common set of signalling pathways. Genetic variants in and expression of individual TNF superfamily cytokines, receptors and signalling proteins have been associated with autoimmune and inflammatory diseases, but their interconnected biology has been largely unexplored.MethodsWe took a hypothesis-driven approach using available genome-wide datasets to identify genetic variants regulating gene expression in the TNF superfamily cytokine signalling network and the association of these variants with autoimmune and autoinflammatory disease. Using paired gene expression and genetic data, we identified genetic variants associated with gene expression, expression quantitative trait loci (eQTLs), in four peripheral blood cell subsets. We then examined whether eQTLs were dependent on gene expression level or the presence of active enhancer chromatin marks. Using these eQTLs as genetic markers of the TNF superfamily signalling network, we performed targeted gene set association analysis in eight autoimmune and autoinflammatory disease genome-wide association studies.ResultsComparison of TNF superfamily network gene expression and regulatory variants across four leucocyte subsets revealed patterns that differed between cell types. eQTLs for genes in this network were not dependent on absolute gene expression levels and were not enriched for chromatin marks of active enhancers. By examining autoimmune disease risk variants among our eQTLs, we found that risk alleles can be associated with either increased or decreased expression of co-stimulatory TNF superfamily cytokines, receptors or downstream signalling molecules. Gene set disease association analysis revealed that eQTLs for genes in the TNF superfamily pathway were associated with six of the eight autoimmune and autoinflammatory diseases examined, demonstrating associations beyond single genome-wide significant hits.ConclusionsThis systematic analysis of the influence of regulatory genetic variants in the TNF superfamily network reveals widespread and diverse roles for these cytokines in susceptibility to a number of immune-mediated diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0329-5) contains supplementary material, which is available to authorized users.

Regulation of vascular endothelial growth factor-C by tumor necrosis factor-α in the conjunctiva and pterygium.

Vascular endothelial growth factorC(VEGF-C) plays an important role in the development of a pterygium through lymphangiogenesis. We examined the association between VEGF-C and tumor necrosis factor-α(TNF-α) in the pathogenesis of pterygia. Cultured conjunctival epithelial cells were treated with TNF-α, and the gene expression levels of VEGFC were evaluated by quantitative polymerase chain reaction(qPCR) and VEGF-C protein expression levels were measured using an enzyme-linked immunosorbent assay(ELISA). In addition, using ELISA, we evaluated the VEGF-C protein expression in the supernatants of cultured conjunctival epithelial cells, in which we neutralized TNF-α using anti‑TNF-α antibody. The gene expression of tumor necrosis factor receptor superfamily, member1A(TNFRSF1A), known as TNF receptor1(TNFR1), was confirmed using reverse transcription PCR in cultured conjunctival epithelial cells. Immunofluorescence microscopy was used to examine the localization of VEGF-C and TNFR1 in pterygium tissues and TNFR1 expression in cultured conjunctival epithelial cells. Immunohistochemistry was used to examine the localization of TNFR1 in pterygia and normal conjunctival tissues. VEGFC gene expression increased in cultured conjunctival epithelial cells 24h after the addition of TNF-α. The secretion of VEGF-C protein was significantly increased 48h after the stimulation of cultured conjunctival epithelial cells with TNF-α. Increased VEGF-C protein secretion stimulated by TNF-α was significantly reduced by anti-TNF-α neutralizing antibody treatment. In cultured conjunctival epithelial cells, TNFRSF1A and TNFR1 were expressed. TNFR1 was immunolocalized in normal conjunctival tissues and in human pterygium tissues as well as in VEGF‑C‑positive epithelial cells from human pterygia. Our data demonstrate that TNF-α mediates VEGF-C expression, which plays a critical role in the pathogenesis of pterygia.

Increased TNFR1 expression and signaling in injured peripheral nerves of mice with reduced BACE1 activity

Hematogenous macrophages remove myelin debris from injured peripheral nerves to provide a micro-environment conducive to axonal regeneration. Previously, we observed that injured peripheral nerves from Beta–site APP Cleaving Enzyme 1 (BACE1) knockout (KO) mice displayed earlier influx of and enhanced phagocytosis by macrophages when compared to wild-type (WT) mice. These observations suggest that BACE1 might regulate macrophage influx into distal stumps of injured nerves. To determine through which pathway BACE1 influences macrophage influx, we used a mouse inflammation antibody array to assay the expression of inflammation-related proteins in injured nerves of BACE1 KO and WT mice. The most significant change was in expression of tumor necrosis factor receptor 1 (TNFR1) in the distal stump of injured BACE1 KO nerves. Western blotting of protein extracts confirmed increased expression of TNFR1 and its downstream transcriptional factor NFκB in the BACE1 KO distal stumps. Additionally, treatment of WT mice with a BACE1 inhibitor resulted in increased TNFR1 expression and signaling in the distal stump of injured nerves. Exogenous TNFα increased nuclear translocation of p65 NFκB in BACE1 KO tissue and cultured fibroblasts compared with control WT. BACE1 regulates TNFR1 expression at the level of gene expression and not through proteolytic processing. The accelerated macrophage influx in injured nerves of BACE1 KO mice correlates with increased expression and signaling via TNFR1, indicating a link between BACE1 activity and TNFR1 expression/signaling that might contribute to repair of the injured nervous system.

Monocyte Recruitment after High-Intensity and High-Volume Resistance Exercise

The innate immune response is generally considered to have an important role in tissue remodeling after resistance exercise. The purpose of this study was to compare changes in markers of monocyte recruitment after an acute bout of high-intensity (HVY) versus high-volume (VOL) lower-body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 yr, 90.1 ± 11.3 kg, 176.0 ± 4.9 cm) performed each protocol in a randomized, counterbalanced order. Blood samples were collected at baseline, immediately (IP), 30 min (30P), 1 h (1H), 2 h (2H), and 5 h (5H) postexercise. Plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), myoglobin, and cortisol were measured via assay. Tumor necrosis factor receptor 1 (TNFr1), macrophage-1 antigen (cluster of differentiation 11b [CD11b]), and C-C chemokine receptor 2 (CCR2) expression levels were measured using flow cytometry. TNFr1 and CD11b were assessed on CD14CD16 monocytes, whereas CCR2 was assessed on CD14 monocytes. Plasma myoglobin concentrations were significantly greater after HVY compared with VOL (P < 0.001). Changes in plasma TNF-α, MCP-1, and expression levels of CCR2 and CD11b were similar between HVY and VOL. When collapsed across groups, TNF-α was significantly increased at IP, 30P, 1H, and 2H (P values < 0.05), whereas MCP-1 was significantly elevated at all postexercise time points (P values < 0.05). CCR2 expression on CD14 monocytes was significantly lower at IP, 1H, 2H, and 5H (P values < 0.05). CD11b expression on CD14 CD16 was significantly greater at IP (P < 0.014) and 1H (P = 0.009). TNFr1 expression did not differ from baseline at any time point. Plasma cortisol concentrations did not seem to be related to receptor expression. Results indicate that both HVY and VOL protocols stimulate a robust proinflammatory response. However, no differences were noted between resistance exercise training paradigms.

NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.

TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) has been reported to induce keratinocyte apoptosis in vitro by engaging its sole receptor of fibroblast growth factor-inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the growth of psoriatic keratinocytes that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary keratinocytes were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by keratinocytes in vitro, which expressed TNF receptor 2 predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein 2 and cellular FLICE-inhibitory protein in lesional keratinocytes. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of keratinocytes under psoriatic inflammation. The activation of nuclear factor-κB signalling-dependent anti-apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in keratinocytes under psoriatic inflammation.

Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

Tumour necrosis factor receptor I blockade shows that TNF-dependent and TNF-independent mechanisms synergise in TNF receptor associated periodic syndrome.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb.

A Model of Post-Infection Fatigue Is Associated with Increased TNF and 5-HT2A Receptor Expression in Mice.

It is well documented that serotonin (5-HT) plays an important role in psychiatric illness. For example, myalgic encephalomyelitis (ME/CFS), which is often provoked by infection, is a disabling illness with an unknown aetiology and diagnosis is based on symptom-specific criteria. However, 5-HT2A receptor expression and peripheral cytokines are known to be upregulated in ME. We sought to examine the relationship between the 5-HT system and cytokine expression following systemic bacterial endotoxin challenge (LPS, 0.5mg/kg i.p.), at a time when the acute sickness behaviours have largely resolved. At 24 hours post-injection mice exhibit no overt changes in locomotor behaviour, but do show increased immobility in a forced swim test, as well as decreased sucrose preference and reduced marble burying activity, indicating a depressive-like state. While peripheral IDO activity was increased after LPS challenge, central activity levels remained stable and there was no change in total brain 5-HT levels or 5-HIAA/5-HT. However, within the brain, levels of TNF and 5-HT2A receptor mRNA within various regions increased significantly. This increase in receptor expression is reflected by an increase in the functional response of the 5-HT2A receptor to agonist, DOI. These data suggest that regulation of fatigue and depressive-like moods after episodes of systemic inflammation may be regulated by changes in 5-HT receptor expression, rather than by levels of enzyme activity or cytokine expression in the CNS.

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1.

TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effects of glucocorti-coids are mediated by induction of this miR, resulting in reduced TNFR1 expression.

The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway

IntroductionMutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations.MethodsHEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively.ResultsTNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients’ data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation.ConclusionsThe novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0604-7) contains supplementary material, which is available to authorized users.

TNFR1 and TNFR2 Expression and Induction on Human Treg Cells from Type 1 Diabetic Subjects

Several autoimmune diseases are marked by a deficiency of soluble tumor necrosis factor (TNF). The TNF deficiency is caused in at least one autoimmune disease, multiple sclerosis, by an overabundance of TNF receptor 1 (TNFR1). Excess TNFR1 binds and inactivates TNF and this leaves less TNF bioavailable. This study sought to determine if expression of fresh or IL2-stimulated TNF receptors on Tregs cells, an important immunoregulatory cell involved in autoimmunity, is altered in type I diabetes. Standard fluorescence analysis was used to examine the levels of TNFR1 and TNFR2 on human Tregs in patients with type I diabetes (T1D) or controls. Fresh Tregs from T1D compared to control Tregs had identical levels of TNFR1. In marked contrast, Type 1 diabetic patients Treg cells had statistically elevated levels of TNFR2 compared to controls. Tregs stimulated with IL2 from both T1D and controls showed marked increase of TNFR2 expression in a dose-response manner, but the dose response increase in TNFR2 was significantly higher for T1D Treg cells. No IL2 dose-response was present for TNFR1 on either T1D or control Tregs exposed to IL2. A large study of serum for secreted levels of TNFR2 also revealed elevated circulating levels consistent with the elevated surface expression on Tregs. These findings suggest that abnormal regulation of TNFR2 expression with elevated cellular and secreted levels of TNFR2 is a characteristic of Type 1 diabetes. It is possible that the relative deficiency of TNF in type I diabetes, in contrast to multiple sclerosis, is caused by excess expression of TNFR such as TNFR2, a binding structure for inactivating TNF.